Human Cellular Models with Biosensors

ABSTRACT

This invention provides biosensors, cell models, and methods of their use for monitoring ionic or voltage responses to contact with bioactive agents. Biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest. Model cells expressing the biosensors can be contacted with putative bioactive agents to determine possible activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of a prior U.S. Provisional Application No. 61/873,950, Human Cellular Models with Biosensors, by Angela Huang, filed Sep. 5, 2013. The full disclosure of the prior application is incorporated herein by reference.

FIELD OF THE INVENTION

The present inventions are in the field of methods and compositions for active agent screening, monitoring of electrical impulses, and for monitoring signal transduction. The inventions present compositions for detection of specific bioactivity, e.g., in targeted regions of representative host cells. Methods include transformation of reprogrammed human cells with targeted sensor constructs specific for, e.g., detection of changes in the ionic environment or voltage potential in a cell.

BACKGROUND OF THE INVENTION

Detection of voltage potentials and changes to the internal ionic environment of cells can be useful in monitoring bioactivities of cells. For example, many cells experience significant changes in internal calcium ion (Ca²⁺) concentration in response to binding of a ligand to a G-protein receptor. In another aspect, certain cells experience large changes in voltage potential across membranes, e.g., in response to contact with neurotransmitters at a synapse. Such cellular changes are responsible for important functions in cells and can be indicative of the health, function, or development processes of the cells.

Genetically encoded calcium indicators (GECI) have been developed to allow general monitoring of the Ca²⁺ concentration in cells. See, e.g., Looger (U.S. 2012/0034691), wherein a calmodulin peptide sequence is combined in a construct with a green fluorescent protein (GFP) reporter. Conformational changes in the calmodulin peptide in response to Ca²⁺ binding changes the efficiency of the GFP fluorescence, resulting in a change in the emissions profile and intensity. Cells transformed with the GECI can be monitored generally for changes in internal Ca²⁺ concentration, e.g., in response to signaling ligands or inhibitors. Also see Griesbeck (U.S. 2009/0035788) wherein a FRET donor and acceptor are separated by a troponin peptide sequence, resulting in a fluorescence change on binding of Ca²⁺. However, such GECIs are limited in their resolution of signal, limited in ability to penetrate multi-cell/3D structures, and in the range of available applicable cell types. Typically, the old art systems are directed to two dimensional microscopic detection of signals from a cell monolayer.

In many cases, signal transduction studies are carried out in cell types that are not representative of the actual cells of interest. For example, researchers may be limited to studying signaling agents and potential therapeutics in rodents or immortal cell lines in vitro, which often provide results not repeated in human cells, or clinical patients. For example, researchers can create host cells for study by introduction of oncogenes to primary cell lines, e.g., with differentiation to a cell type of choice. However, after this process history, the cells cannot be relied on to respond normally on contact with bioactive agents. Weiss (U.S. Pat. No. 7,101,709) discloses methods of preparing multipotent mouse neural stem cells. The cells could be differentiated and transplanted to somewhat immunoprivileged CNS locations. Again, the cells would be non-representative for many studies, and signal detection is limited to immunochemical means.

In view of the above, a need exists for model cell systems representative of cells and tissues existing in live animal systems of interest. It would be desirable to have sensor peptide constructs that can be targeted to specific intracellular locations. Benefits could be realized if systems were available allowing three dimensional signal detection in mock tissues of representative cells in vitro. The present invention provides these and other features that will be apparent upon review of the following.

SUMMARY OF THE INVENTION

The present biosensors combine complementary features to detect intracellular changes associated with signaling in vivo. The present biosensors find utility in a variety of contexts. For example, the biosensors in human iPSCs-derived cell types can function to image 2D or 3D cellular models of in cells expressing certain pathologies. Such models can be useful in screening and evaluation of candidate drug compounds for chemical, biologic, therapeutic, and toxicological effects.

The synthetic and genetically encoded biosensors can function as reporters of calcium (Ca²⁺) concentrations in human cells. The biosensors can have structures targeting cellular compartments, e.g., such as the nucleus, cytoplasm, plasma, certain membrane surfaces, and/or the like. The biosensors can optionally function as reporters of cellular voltage changes in human cell compartments. For example, voltage sensors can evaluate and report fluctuations in membrane potentials due to differentials or changes in sodium (Na⁺) and potassium (K⁺) concentrations.

The biosensors can be configured to function in a cell-type specific manner. For example, biosensors can be genetically modified to contain promoter sequences specific to certain cell types, e.g., dopaminergic neurons, GABAergic neurons, astrocytes, cardiomyocytes, immortalized human cancer cell lines, HSCs, NPCs, human cells in general and/or MSCs.

In one embodiment, the biosensor is a voltage sensor. The voltage sensor can include, e.g., a peptide construct comprising a transmembrane domain, voltage sensitive domain, and a reporter domain. For example, the voltage sensor construct can include a transmembrane domain adapted to integrate into a membrane of a human cell, a voltage sensing domain sensitive to H⁺, Na⁺ and/or K⁺ concentration, and a fluorescent reporter domain. The fluorescent reporter can be adapted to fluoresce at wavelengths in the range from 500 nm to 750 nm. The biosensor can be adapted to change conformation (e.g., in the voltage sensing domain) in response to local voltage potentials, resulting in the fluorescent reporter domain changing fluorescent emission characteristics. In many embodiments, the transmembrane domain and voltage sensing domain are different domains (e.g., having different non-homologous sequences, or being derived from different parent peptides).

In certain embodiments of the voltage sensor system, the voltage sensor peptide includes a voltage sensing domain comprising a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVH WNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGVDDGRMEIPATWWN KLRKVISPFVMSFGFRVFGVVLIIVDFVLVIVDLSVTDKSSGATTAISSI SLAISFFFLIDIILHIFVEGFSQYFSSKLNIFDAAIVIVTLLVTLVYTVL DAFTDFSGATNIPRMVNFLRTLRIIILVRIIILVRILRLASQKTISQN. In preferred embodiments, the voltage sensor peptide sequence retains I123, R220, R226, R229, R235 and/or R238 residues. In a more preferred embodiment, the voltage sensor domain peptide wherein the sequence retains at least R235 and I123.

In some embodiments of the voltage sensor system, the fluorescent reporter domain peptide includes a fluorescent domain comprising a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAK LKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED.

In certain embodiments, the voltage sensor peptide construct includes the peptide sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to each of SEQ ID NO: 1 and SEQ ID NO: 2.

Another inventive aspect includes a nucleic acid construct encoding any of the voltage sensors described herein. Further, the nucleic acid construct can comprise a tag sequence selected from the group consisting of: a nucleus localization signal (NLS) tag, a mitochondrial localization tag, and a ciliary tag. Further, the nucleic acid construct can “ ” include the NLS tag comprising at least 80% identity to the peptide sequence:

(SEQ ID NO: 3) DPKKKRKV.

The inventive contributions of the present application include a human cell comprising the voltage sensor described herein. In preferred embodiments, the human cell is an iPSC derived cell. For example, the cell can be derived from induction of a fibroblast or a blood cell to a pluripotent or immortal status. In many cases, the cell is derived from a human patient derived cell type.

The biosensors of the invention include calcium sensor. For example, a calcium sensor peptide construct can include a calcium binding domain, a EF-hand troponin-like binding domain, and fluorescent reporter domain. Often the fluorescent reporter is adapted to emit at wavelengths in the range from 500 nm to 750 nm. The fluorescent reporter domain can be adapted to change fluorescent emissions characteristics, e.g., in response to conformational changes in the EF-hand troponin-like domain.

The calcium sensor peptide construct can include a calcium binding domain comprising a calmodulin peptide sequence. For example, the calcium sensor peptide construct can include a calcium binding domain is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 4) EFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSLGQN PTEAELQDMINEVDADGDGTFDFPEFLTMMARKMMNDTDSEEEGVQGTSE EEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIEDLMKDSDKNN GRIDFGEKLTDEEV.

The calcium sensor peptide construct can include a EFhand binding domain is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 5) FKEAFSLFDKDGDGTITTKELGTVMRSL-ELDAIIEEVDEDGSGTIDFEE FLVMMVRQ.

The calcium sensor peptide construct can include a fluorescent reporter domain is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAK LKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED.

The calcium sensor peptide construct can includes the peptide sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to each of SEQ ID NO: 4 and SEQ ID NO: 5.

Another inventive aspect described herein includes nucleic acid constructs encoding any of the calcium sensors described herein. Further, the nucleic acid construct can comprise a tag sequence. For example, the tag sequence can include a targeting sequence, such as a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, and/or a ciliary tag.

The inventive contributions of the present application include a human cell comprising any calcium sensor described herein. In preferred embodiments, the human cell is an iPSC derived cell. For example, the cell can be derived from a fibroblast or a blood cell.

The inventions include methods of reprogramming and monitoring cells. For example, in a method of reprogramming fibroblasts can include transforming the fibroblasts with one or more constructs comprising a human clock gene and human Bmal1/2/3/4 genes having E-box promoters, synchronizing the circadian rhythm of the fibroblasts, modifying transcriptional regulatory control of the fibroblasts (thereby converting them into inducible pluripotent stem cells), and reprogramming the stem cells into inducible neurons (iN), glial cells (astrocytes included, iG), inducible pluripotent stem cells (iPSCs), or inducible neural progenitor cells (iNPCs). In many of the methods for reprogramming using circadian rhythms of human cell types, the transforming construct comprises a nucleic acid encoding a voltage sensor described herein or calcium sensor described herein.

The methods can include, e.g., modifying transcriptional control by providing specific transcription factors suitable for a lineage of the fibroblasts. The transcription factors can include the factors specific for a cellular lineage of the fibroblasts modified to include a circadian regulatory element (E-box promoters, an artificial E-box-like promoter sequence tag, a chemical agent that alters or synchronizes circadian rhythms cycles, or a synthetic transcriptional enhancer element) for reprogramming using circadian rhythms of human cell types.

The cellular composition can further include the co-culturing iN cells and iG cells to create a 3D model on a scaffold in vitro. Also, human cancer cell lines and cancer stem cells can be cultured in 3D spheroid manners using standard culturing hardware and conditions. Biosensor constructs of the invention can be used to image the resultant tissues and monitor changes in the cell voltage potentials and/or calcium levels, e.g., in real time.

Heme-binding and ATP-binding biosensors can be designed using similar logic as the calcium and voltage biosensors described herein. Both biosensors are genetically encoded, with specific domains for binding to heme molecules or ATP, respectively. These biosensors are attached to a fluorescent reporter (600 nm-700 nm range), whose intensities are modulated due to conformational changes due to domain binding. In alternatives to a single fluorescent or intensiometric reporter, the biosensor domains can be attached to a pair of FRET fluorescent reporters of various wavelengths and become a FRET sensor.

The present inventive methods include compositions and techniques for screening agents that influence the calcium or voltage potential of cells. For example, methods for screening active agents can include transforming one or more cells with a nucleic acid encoding a voltage sensor or calcium sensor, expressing the voltage sensor or calcium sensor in the cells, contacting the cells with candidate active agents, and detecting a change in florescence of the voltage sensor or calcium sensor in response to the agent. Typical agents can include, e.g., members of a small molecule chemical library. For example, the agents can be reviewed for an activity resulting from interactions with GPCR, a membrane channel, a receptor, and/or associated signaling pathways.

It is further envisioned that the methods are useful in imaging live cells in low, medium, and/or high throughput assay formats. For example, cells in 3D arrays, transformed and expressing biosensor constructs of the invention can be viewed in real time, e.g., using microscopic imaging systems. Optionally, biosensor cells in surface or suspension culture can be monitored using confocal imaging, FACS sorters, CCD video imaging, and/or the like.

3D human cancer cell models have been used as an improved predictor of tumor responses to drug candidates using library screening high throughput screening (HTS) methods. Cells used for such studies aren't limited to immortalized cancer cell lines. Cancer stem cells, primary tissue cells, and 3D printed tissue types can be used for screening.

DEFINITIONS

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a surface” includes a combination of two or more surfaces; reference to “bacteria” includes mixtures of bacteria, and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

iPSC refers to inducible pluripotent stem cells; iN refers to inducible neurons; iG refers to inducible glial cells (including astrocytes); and iNPCs inducible neural progenitor cells.

Near-Infrared refers to light wavelengths ranging from about 600 nm to about 1400 nm.

The term “conservative variant” includes modifications of given sequences that result in conserved function. For example, in the context of nucleic acids, owing to the degeneracy of the genetic code, “silent substitutions” (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid.

Similarly, conservative variants in the context of peptide sequences can be expected to retain function. For example, Guo, “Protein Tolerance to Random Amino Acid Change”, (PNAS 101:9205-10; 2004), demonstrates that one of skill can modify peptides successfully even “without detailed knowledge of the ways in which a protein's structure relates to its functional usefulness . . . ” Guo finds only 25% of random mutations lead to substantial loss of activity. Guo extensively discusses how one of skill can take into consideration active site location, alpha helices, beta sheets, hydrophobic interactions, turns and loops, conserved sites and the like to intelligently avoid loss of activity, e.g., by substitution avoidance at key positions or with conservative amino acid substitutions. Further, Guo states that his “database can be a valuable resource for predicting the effects of mutations on protein function . . . .” Substitutions to known structures are predictable and in possession the of those having the structural information. Therefore, conservative amino acid substitutions, in which one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed constructs, and expected to retain function. One of skill will recognize that individual substitutions, deletions, or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Thus, “conservative variations” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.

TABLE 1 Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chart wherein TempoCal™ has been incorporated into human cells and stimulated with histamine (20 μM), carbachol (20 μM), and ionomycin (20 μM) for an average response 9-12 fold over control. Histamine: a known substance that stimulates intracellular calcium increases. Carbachol: a known medication to treat glaucoma; an anti-apoptosis agent, aCHR agonist, and TNF-alpha inhibitor. Ionomycin: an ionophore used in research to raise intracellular levels of calcium.

FIG. 2 shows a chart wherein TempoMito™-containing cells have been stimulated with histamine (20 μM), and carboxyamidotriazole (20 μM). Histamine: a known substance that stimulates intracellular calcium increases. Carboxyaminoimidazole (CAI): an orally-active agent, non-voltage-operated calcium channel blocker.

FIG. 3 shows TempoVol™-incorporated cells respond to pH 3, 0.1M CaCl₂, EDTA, and Ca²⁺ Mg²⁺ Free solutions.

FIG. 4 shows TempoCal™, TempoMito™, and TempoVol™ cellular responses from a single 2.5 mM CaCl₂ stimulation.

FIG. 5 shows TempoVol™: cellular responses to pH3, 1.0 mM CaCl₂, 10 mM CaCl₂, and 10 mM EDTA.

FIG. 6 shows TempoMito™ responses using U2-OS human osteoblastoma cells.

FIG. 7 shows Tempo's iPS derived human neural progenitor cells. Immunohistochemistry employs biomarker vimentin antibodies. Human vimentin is an intermediate filament type and is expressed by neural progenitor/stem cells of the central nervous system.

FIG. 8 shows Tempo's iPS derived human neural progenitor cells in a light transmission microscopic field.

FIG. 9 shows TempoCal™ biosensor expressed in Tempo's iPS derived human neural progenitor cells.

FIG. 10 shows Tempo's iPS derived human astrocyte cells in a light transmission view.

FIG. 11 shows Tempo's iPS derived human astrocyte cells. Immunohistochemistry employs biomarker GFAP antibodies. GFP (glial filament protein) is a class-III intermediate filament and a cell specific marker that distinguishes astrocytes from other glial cells during the development of the central nervous system.

FIG. 12 shows the TempoCal™ biosensor expressed in Tempo's iPS derived human astrocyte cells.

FIG. 13 shows the TempoMito™ biosensor expressed in Tempo's iPS derived human astrocyte cells.

FIG. 14 shows the TempoMito™ biosensor expressed in Tempo's iPS derived human neural progenitor cells.

FIG. 15 shows the TempoCal™ biosensor expressed in Tempo's proprietary neuroepithelial cells.

FIG. 16 shows the TempoMito™ biosensor expressed in Tempo's proprietary neuroepithelial cells.

FIG. 17 shows Tempo's iPS derived human oligodendrocyte progenitor cells. Immunohistochemistry employed biomarker O4 antibodies.

FIG. 18 shows Tempo's iPS derived human oligodendrocyte progenitor cells induced with T3 hormone (70 ng/ml) after 6 days Immunohistochemistry employs biomarker myelin basic protein (MBP) antibodies.

FIG. 19 shows the TempoVol™ biosensor expressed in Tempo's proprietary neuroepithelial cells.

FIG. 20 shows Tempo's iPS derived human dopaminergic neurons. Immunohistochemistry employs biomarker Thymine Hydroxylase (TH) antibodies.

FIG. 21 shows Tempo's immortalized neuroepithelial cells tested as a 3D spheroid model using Lipidure-coated 96-well U-shaped plates. Diameter of spheroid shown=250 μm.

DETAILED DESCRIPTION

The present invention includes biosensor constructs, reprogramming methods to prepare cells useful in receiving the constructs, and assay methods employing the biosensors, e.g., to detect the presence of active agents or signals in living cells.

The biosensors generally include various complementary domain structures working together to sense a voltage or ion at a particular intracellular location, and to provide a distinct signal correlated to changes in the parameter. In a particularly useful embodiment, the biosensors are expressed in cells representative of a particular species (e.g., human), cell type of interest, or in a cell expressing a pathology of interest. Cells expressing the biosensors can be exposed to conditions or agents and monitored for signals indicating cell responses.

Biosensors Generally.

Biosensors of the present invention are generally engineered to include components specialized in providing, e.g., location, sensing, and reporting. In expression of a sensor nucleic acid construct, the peptide product can be positioned within the cell, e.g., by chaperone or transit sequences, hydrophobic affinities, or ligand/receptor interactions. The sensing domains typically change conformation in response to a changed intracellular condition or binding of a signal ion or molecule. Reporting domains typically present a detectable signal that changes in response to conformational changes in the associated sensing domain.

The intracellular location of a biosensor can optionally be controlled by a targeting domain or “tag”. In some biosensors, intracellular location is generalized or passively determined. For example, the sensor may be generally dispersed throughout the nucleus and/or cytoplasm. In other cases, the function or specificity of the biosensor signal may depend on localization at a particular intracellular membrane or organelle. Examples biosensor tags include hydrophobic peptides directed to interact with membranes, ionic peptides directed to disperse in cytoplasm, chaperone sequences directing the biosensor to a particular compartment, and/or ligands directed to receptors. Tags useful in the study of intracellular signaling and in agent screening assays can include, e.g., NLS tags, lipid membrane tag, endoplasmic reticulum (ER) tags, golgi tags, endosome tags, ciliary tags, and/or the like.

Sensing domains in the biosensors change conformation in response to a changed physical condition, binding of a ligand, or change in an ionic environment. The conformational changes can, e.g., cause conformational changes in an associated reporter domain, or reposition the reporter domain to enhance or diminish the signal. Sensing domains can change conformation in response to binding of a peptide, binding of a nucleic acid, interaction with a protease, interaction with a phosphatase, changes in pH, changes in ionic strength, changes in a voltage potential, and/or the like.

Reporter domains of the biosensors can be of any appropriate type known in the art. However, in preferred embodiments, the reporters comprise one or more peptide domains, e.g., so they can be easily employed in in vivo systems. Typically the peptide reporter domain provides a specific fluorescent emission in response to a specific interrogating excitation wavelength of light. In the context of sensor domain conformational changes, FRET strategies can be effective, e.g., wherein the biosensor construct comprises paired donor/acceptor peptide pairs. In certain embodiments, the reporter domain is adapted to provide an emission in the red to near-infrared spectrum, e.g., to allow the signal to pass and be detected in three dimensions through cells or tissue layers. For example, relatively penetrating reporter emission signals can range from 500 nm to 1400 nm, 550 nm to 900 nm, 600 nm to 850 nm, 650 nm to 800 nm, or about 700 nm. In an exemplary embodiment, the reporter domain includes fluorescence enhancing amino acids, such as tryptophan, tyrosine, phenylalanine, which contribute to their intrinsic fluorescence of the domain. Optionally, the reporter domain can include regions naturally modified post-translationally to provide the desired long wavelength emissions. For example, a domain that emits in the red regions can be a modified green fluorescent protein where the fluorophore originates from an internal serine-tyrosine-glycine sequence which is modified to a 4-(p-hydroxybenzylidene)-imidazolidin-5-one structure.

Biosensor constructs can include, e.g., two, three, or four of the above described domain types, in any of a variety of functional configurations. For example, a sensor domain and reporter domain in any order can constitute a biosensor. Often, the biosensors of the invention include at least three domains, e.g., a targeting domain, a sensor domain, and a reporting domain. The three domains can be in any order, but typically the targeting domain is on one end of the construct, the sensor in the middle and the reporter on the second end. In certain configurations, the biosensor construct can be in the order (C-terminal/N-terminal or N-terminal/C-terminal) of: sensor/reporter; targeting/sensor/reporter; targeting/reporter/sensor; targeting/reporter1/sensor/reporter2 (e.g., FRET); reporter1/sensor/reporter2; sensor/reporter1/reporter2; and targeting/sensor/reporter1/reporter2.

Typically, the domains are linked together in a commonly translated construct of a single linear peptide. Optionally, the constructs can include one or more domains not in the same peptide chain as another domain. For example, separate domains may be associated in a non-covalent interaction, such as a hydrophobic interaction, a chelation, a ligand/receptor interaction, an antibody/antigen interaction, and/or the like.

In some cases, a single domain may have more than one function. For example, a sensor domain may also have a structure functioning as a targeting domain. In one embodiment, a domain may have a series of transmembrane domains, acting as both as a sensor (e.g., ligand responsive ion channel) and a membrane specific targeting domain. In another aspect, a sensor domain could also include a reporter function, e.g., acting as a donor or quencher member of a FRET pair with a separate reporter domain.

Voltage Sensing Biosensors.

Voltage sensors of the invention include many of the aspects described above, wherein the sensor domain is responsive to changes in voltage potential. For example, the voltage sensor may be sensitive to electrostatic influences in the local environment, e.g., a voltage differential across a membrane to which it is bound. Optionally the voltage sensor is sensitive to the local ionic environment. In living cells, the voltage potential is usually related to the absolute concentration of H⁺, Ca²⁺, K⁺ and/or Na⁺, or a concentration differential of these ions across a membrane. Therefore, voltage sensor domains typically include domains sensitive to electrostatic forces, H⁺, Ca²⁺, K⁺ and/or Na⁺ concentration. Such sensors are particularly useful in association with relatively electroactive cells such as muscle cells, heart cells, and nerve cells.

Importantly, such voltage sensors can be configured to target and report the ion/voltage status specifically at intracellular locations. For example, the construct can include a targeting domain directing the construct to a cellular compartment or surface such as the nucleus, sacroplasma, plasma membrane, nerve axon, cilia, or synapse. Thus, not only can signal transduction be monitored in a cell generally, but more specific voltage differential effects can be visualized at the sub-cellular level.

Typical voltage sensitive biosensors of the invention include a transmembrane domain, voltage sensing domain, and a reporter domain. The transmembrane domain may act as a targeting domain and/or a voltage sensing domain, in some instances. In one embodiment, the voltage sensor has the domains in the order transmembrane/sensor/reporter. In preferred embodiments, the reporter is a fluorescent peptide, e.g., emitting in the red to near-infrared range.

A preferred voltage biosensor construct includes a combination of an ion channel transmembrane peptide, an ion channel voltage sensing peptide domain, and a fluorescent peptide. In an exemplary embodiment, the voltage sensor includes a modified transmembrane peptide, a modified voltage, and a fluorescent protein modified to provide red emissions. The domains of the constructs are typically configured from a combination of bioinformatics/database sequences as modified evolutionary mutagenesis.

In a particular voltage sensing domain the sequence comprises:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVH WNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGGDDGRMEVPATWWN KLRKVISPFVMSFGFRVFGVVLIIVDFVLVIVDLSVTDKSSGATTAISSI SLAISFFFLIDIILHIFVEGFSQYFSSKLNIFDAAIVIVTLLVTLVYTVL DAFTDFSGATNIPRMVNFLRTLRIIRLVRIIILVRILRLASQKTISQN.

Conservative variations of the sequence would be expected to retain substantial useful function. The present voltage sensor domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1. Function is expected to be best preserved with conservative substitutions and if the sequence retains at least 1, 2, 3, 4, 5, or all 6 of the following amino acid residues: I123, R220, R226, R229, R235, or R238. In many embodiments, preservation of R235 and I123 can be particularly useful in retaining optimal voltage sensor function.

In a particular reporter domain for the voltage sensor peptide, the sequence comprises:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAK LKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED. Conservative variations of the sequence would be expected to retain substantial useful function. The present fluorescent reporter sensor domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 2.

Preferred voltage sensing biosensors of the invention include both the voltage sensor of SEQ ID NO: 1 and the fluorescent reporter of SEQ ID NO: 2, and/or their conservative variants, as discussed above.

Calcium Ion Sensing Biosensors.

Calcium sensor constructs of the invention include many of the aspects described above for biosensors generally, but the sensor domain is responsive to changes in calcium ion levels. For example, the calcium sensor will typically bind Ca²⁺ with a certain affinity and change conformation to some degree depending on the local Ca²⁺ concentration. Ca²⁺ can vary dramatically depending on cell type, and according to the influence of induced signals. For example, it can be informative to monitor muscle cells, nerve cells, cells responding to g-protein controlled signals, cells undergoing apoptosis, and/or the like. In living cells, Ca²⁺ levels often vary with intracellular locations. The present calcium sensors can include targeting domains directing the sensors to any intracellular compartment of membrane, such as, e.g., a vacuole, the nucleus, cytoplasm, synapse, endoplasmic reticulum, and/or the like.

Calcium sensor domains are typically peptides homologous to portions of one or more calcium binding proteins. For example, a calcium binding domain can have a sequence similar to an evolutionary sequence found in calmodulin, calexcitin, parvalbumin, S100 proteins, calcineurin, and/or the like. All that is necessary for the sensor role is that the peptide, or peptide fragment, changes conformation with changes in calcium concentration. The change in conformation will translocate the reporter group and typically change the emission profile or intensity. Modern protein engineering techniques can be used to engineer enhancements causing the translocation of the reporter to be enhanced or quenched, e.g., by induced contact or induced conformational changes in the reporter itself.

A preferred calcium biosensor construct includes a combination of a tandem array of calcium binding domains (namely, EFhand domains) that include calmodulin and troponin motifs, and a fluorescent peptide. In an exemplary embodiment, the calcium sensor includes a modified calcium binding domain from calmodulin, troponin, and a fluorescent protein modified to provide red emissions. The domains of the constructs are typically configured from a combination of bioinformatics/database sequences as modified evolutionary mutagenesis.

In a particular calcium binding domain the sequence comprises:

(SEQ ID NO: 4) EFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSL GQNPTEAELQDMINEVDADGDGTFDFPEFLTMMARKMMNDTDSEEEG VQGTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIED LMKDSDKNNGRIDFGEKLTDEEV. Conservative variations of the sequence would be expected to retain substantial useful function. The present calcium binding domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4.

In a particular EFhand domain for the calcium sensor peptide, the sequence comprises:

(SEQ ID NO: 5) FKEAFSLFDKDGDGTITTKELGTVMRSL--ELDAIIEEVDEDGSGT IDFEEFLVMMVRQ. Conservative variations of the sequence would be expected to retain substantial useful function. The present EFhand domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 5.

In a particular reporter domain for the calcium sensor peptide, the sequence comprises:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQ TAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPE GFTWERVTRYEDGGVVTVMQDTSLED. Conservative variations of the sequence would be expected to retain substantial useful function. The present fluorescent reporter sensor domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 6.

Preferred calcium sensing biosensors of the invention include one, two, three or more of the domains identified in SEQ ID NOs: 4, 5, 6, and 7, and/or their conservative variants, as discussed above.

Biosensor Constructs in Human Cells.

The biosensors described herein can be incorporated into cells to monitor voltage and ion conditions within the cells. For example, nucleic acid constructs encoding the biosensor peptide domains can be transformed or transfected into eukaryotic cells for expression, e.g., using appropriate promoters, as is known in the art. The cells are preferably human, providing the benefit of a reliable host cell model, e.g., for study of human signal transduction and disease states. The cells are preferably stem cells or cells differentiated to a particular cell type of interest.

The nucleic acid construct can encode any biosensor described herein. For example, the construct can encode a combination of a voltage sensor domain, calcium sensor domain, targeting domain, and/or reporter domain. In one embodiment, the nucleic acid construct includes transient expression vector components directing expression of peptide chains comprising a combination of domains providing a functional biosensor. For example, the nucleic acid can express a single peptide chain comprising a combination of calcium binding domain, troponin domain, and reporter domain. In another example, the nucleic acid construct can encode a peptide comprising at least a combination of a transmembrane domain, a voltage sensing domain, and reporter domain.

In other embodiments of the nucleic acid expression constructs, an expression vector includes sequences encoding peptides of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and/or 7. In certain embodiments functioning as voltage biosensors, the vector can include a combination of nucleic acid sequences encoding the peptides of SEQ ID NOs: 1 and 2, e.g., along with a sequence encoding a transmembrane domain, such as a G-protein domain or an ion channel domain. Such a construct can encode functional peptide sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID No: 1 and/or SEQ ID NO: 2. In preferred embodiments, the construct encodes a homologue of SEQ ID NO: 1 retaining at least the amino acids at I123, R220, R226, R229, R235, and/or R238. In many embodiments, preservation of R235 and I123 can be particularly useful in retaining optimal voltage sensor function. In other embodiments functioning as calcium sensitive biosensors, the vector can include a combination of nucleic acid sequences encoding the peptides of SEQ ID NOs: 4, 5, 6, and/or 7. Such a construct can encode functional peptide sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and/or SEQ ID NO: 7.

Useful nucleic acid constructs of the above expression vectors can include additional sequences encoding targeting tags, such as, e.g., a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, a mitochondrial tag, and/or a ciliary tag. For example, the targeting tag can include a sequence at least 90% identical to a sequence encoding the peptide tag sequence of SEQ ID NO: 3.

The present inventions include cells comprising the biosensors discussed herein. For example, nucleic acid constructs coding the biosensor peptides can be transduced or transfected into eukaryotic cells of choice. In preferred embodiments, the cells originate from a mammal, most preferably from a human. In many embodiments, the cell is an immortalized stem cell, or a cell fully or partially differentiated from a stem cell.

In the context of the present biosensors, cells can be initially derived from human patient samples. The advantage of such cells is that they can provide very representative responses to active agents and changed conditions for that patient. Thus, such cell models are more likely to provide information on the modes of action or efficacy of a candidate therapeutic for that patient. For example, such cell models can aid in the identification of custom tailored treatment for patients with certain disease states, such as autoimmune diseases, neurological diseases, cancer, diabetes, or pathologies from genetic flaws.

Alternately, the models can employ cells harboring a particular non-endogenous gene of interest, introduced by genetic engineering techniques. For example, the gene of interest can encode a receptor molecule, a G-protein coupled receptor, or an ion channel of interest. Typically, the host cell is a cell most representative of the cell type of interest in the research. Such model cells can be useful in providing more representative results, e.g., in monitoring a signal transduction or in screening prospective agents active in the modulation of the gene of interest.

Cellular models can be derived from inducible cells available from primary culture of cells from living animals. For example, fibroblasts or undifferentiated cells from circulating blood can be induced to provide pluripotent cells. It is notable that epigenetic processes can play a key role in directing the status of a cell to stem cell, progenitor cell, or mature cell. In Lister (Nature 471 (7336): 68-73, 2011), aberrant epigenomic programming was found capable of inducing a variety of different pluripotent stem cells (iPSCs). Female lung fibroblasts, adipose cells, and foreskin fibroblasts were reprogrammed into induced pluripotent state using OCT4, SOX2, KLF4, and MYC genes. The iPSCs were found to be similar to embryonic stem cells in many characteristics, including DNA methylation patterns. Such concepts can be used to reprogram cells, e.g., in combination with further circadian synchronization techniques, described below.

In addition to inducement by action of immortalizing genes, cell signaling was found to influence epigenetic processes governing differentiation. In the research of Baylin (e.g., Nature Biotechnology 28 (10): 1033-8, 2010), several signaling pathways were suggested as important in the induction and maintenance of embryonic stem cells and in their differentiation. For example, signaling pathways of growth factors can play a role in epigenetic regulation of cellular differentiation. These growth factors include, e.g., transforming growth factors (TGFs), fibroblast growth factors (FGFs), and bone morphogenetic proteins. Another important factor in induction and differentiation can be the Wnt signaling pathway.

Circadian rhythm influences on cellular synchronization can be employed in inducement of pluoripotential cells. For example, cells can be reprogrammed into inducible pluripotent stem cells using human clock gene and human Bmal1/2/3/4 genes and their E-box promoters. In one aspect, fibroblast iPSCs can be generated by such reprogramming, and further directed to provide inducible neurons (iN), glial cells, or inducible neural progenitor cells (iNPCs), as desired. The reprogramming factor for each cell type is typically a transcriptional regulator that is specific for the cellular lineage. Each factor can be modified to be controlled by a circadian regulatory element (such as, E-box promoters or an artificial E-box-like promoter sequence tag). Such promoter sequences can be added to each transcriptional regulator, thus forming a novel transcriptional element for control regulated by human Clock and Bmal genes.

To complement the biosensor systems described herein, induced cells can be cultured together in a “tissue” structure, e.g., in three dimensions. In this way, the cell to cell contacts of interest can be studied, e.g., using penetrating imaging available in the form of near infrared reporter signals. For example, co-cultures of iN and iG can be prepared to create a 3D model of a neuronal structure. The structure can be further controlled, e.g., using an appropriately structured scaffold, e.g., using materials not opaque to reporter signals, e.g., for confocal microscopic review.

Screening and Assay Methods Using Biosensors in Model Cells

The biosensor constructs, e.g., engineered into model cells of interest, can more predictably provide assay and screening results relevant to life science research and study of clinical pathologies of interest. That is, the present targeted biosensors in appropriate cell types, differentiated to a particular phenotype, can provide models more likely to anticipate a normal response in the modeled organism than, e.g., old art xenotypic models.

The initial steps in preparing a model system can be to identify the cell type of interest and the signal to be detected. For example, to study certain neurological disease states, one may choose to target a voltage sensor to the plasma membrane, e.g., in model cells differentiated from iPS cells. Alternately, one may elect to study a blood cell cancer using a hematopoietic blast induced to a model CML cell and signaling from a calcium sensitive biosensor construct. Typically the biosensor constructs are transiently expressed in the cells using, e.g., a CMV constitutive promoter or a cell-type-specific promoter.

Once the model system is established, the biosensor can be monitored in a single cell, or across an array of cells. For example, a cell can be exposed to a signaling agent to see if the cell type responds to that agent, e.g., a cytokine or candidate small molecule bioactive agent. Optionally, the cell can be co-transfected with a second expression construct of a peptide of interest (e.g., a tumor-associated antigen or oncogene) to monitor any influence of the external gene in a signaling pathway.

In other embodiments, the cells can be segregated into arrays complementary to low, medium, or high throughput assay formats. For example, cells can be dispensed into 96-well plates, onto a micro-well array, or a FACS sorter, for separate exposure to library members of putative candidate agents. Such arrays can be reviewed suing standard fluorescent detection equipment. Optionally, the arrays can be reviewed photographically with digital CCD based cameras. Changes in a signal, e.g., as compared to a positive or negative reference, can be flagged for additional characterization.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention. The inventive concepts disclosed herein include certain biosensors sensitive to intracellular conditions, cells expressing the biosensors, and methods of monitoring the cells to detect intracellular changes, e.g., associated with an activity of an externally applied agent.

Example 1 Voltage Sensor Construct

Presented is a voltage sensor for use in intracellular environments. The voltage sensor is a peptide construct featuring a combination of interacting structural features. The expressed peptide construct includes a transmembrane domain, voltage sensing domain, and a fluorescent reporter. The transmembrane can anchor the sensor to a lipid membrane in a cell of choice. The voltage sensing domain can include, e.g., polar or ionic groups that are sensitive to changes in the surrounding ionic environment or to a voltage potential across the anchoring membrane. The fluorescent reporter is a fluorescent peptide sequence, e.g., adapted to be sensitive to conformational changes in other domains of the construct.

The transmembrane domain typically includes hydrophobic amino acid residues that interact with lipids of the membrane to anchor the construct. Further, the transmembrane domain can provide multiple transmembrane structures, together comprising an ion channel or voltage-gated channel.

The voltage sensing domain of the exemplary embodiment includes the following peptide sequence:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVK KVHWNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGGDDGRMEV PATWWNKLRKVISPFVMSFGERVEGVVLIIVDEVLVIVDLSVTDKSS GATTAISSISLAISEFFLIDIILHIFVEGESQYFSSKLNIFDAAIVI VTLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIIILVR ILRLASQKTISQN.

Structural features of note in the voltage sensing sequence include a low complexity region (amino acids 63-75), transmembrane domain (amino acids 109-131), and ion transport (amino acids 149-245). Particularly important residues in the conformational response action include I123, R220, R226, R229, R235, R238; particularly residues R235 and I123.

In this construct, the far-red long wavelength properties fluorophore red color intensities should be particularly sensitive to any changes in residues R235 and I123. Conformational changes in the sensing and transmembrane domains (e.g., particularly in a membrane anchored environment) lead to substantial measurable changes in the intensity (ΔF) and spectrum contours of the fluorophore domain emissions.

The following is a sample of a useful fluorescent-red reporter domain sequence:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQ TAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPE GFTWERVTRYEDGGVVTVMQDTSLED.

Example 2 Calcium Sensor Construct

The structural features of exemplary calcium sensor constructs include a complementary combination of a calmodulin-binding domain, a troponin domain, and a fluorescent reporter domain. The construct can further include one or more tag sequences to target the construct to a particular intracellular location or environment. The complementary combination of tandem arrays of EF-hand domains that include a calmodulin-like-binding domain and a troponin-like domain, and a fluorescent reporter domain. The fluorescent reporter is a fluorescent peptide sequence, e.g., adapted to be sensitive to conformational changes in other domains of the construct.

The calcium sensor components interact as follows. Calcium-binding to EF hand domains leads to a conformational change and surface hydrophobicity changes in the peptide construct. The changed calcium binding domains then interact differently with the fluorescent reporter domain causing a substantial and measurable change in the intensity of the fluorophore (ΔF) emissions.

The calcium sensing domains of the exemplary embodiment includes the following peptide sequence:

(SEQ ID NO: 12) MVDSSRRKWNKAGHAVRAIGRLSSPVVSERMYPEDGALKSEIKK GLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNED YTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEENNMAVIK AEFMRFKEHMEAGSVNGHEFEIAEGEGEGRPYEAGTQTARLKVT KGGPLPFAWDAILSPQIMYGSAKAYVKHPADIAPDYLKLSFPEA GFKWERVMNFEDGGVVHVNQADSSLQDGVFIAYKVKLRGTNFAP PDGPVMQKKATMGWEATRDQLTEEEFRASFNHFDRDHSGTLGPE EFKACLISLDHMVLLTTKELGTVMRSLGQNPTEAELQDMINEVD ADGDGTHOPEFLTMMARKMMNDTDSEEEGVQGTSEEEELANCFR IFDKDANGFIDEELGEILRATGEHVTEEDIEDLMKDSDKNNGRI DFGEKLTDEEVFKEAFSLFDKDGDGTITTKELGTVMRSLELDAI IEEVDEDGSGTIDFEEFLVMMVRQGQNPTKEEELANCFRIFDKN ADGFIDIEELGEILRAT.

The red fluorescent indicator can include the following far-red reporter domain sequence:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQT AKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGF TWERVTRYEDGGVVTVMQDTSLED.

Example 3 Targeting Tags

In many cases, it is advantageous to direct the voltage sensor or calcium sensor to a particular intracellular membrane or compartment. The biosensors of the invention can include peptide segments adapted to have an affinity for a cellular target.

Exemplary peptide sequences useful in targeting biosensors to a desired intracellular location include, e.g.:

NLS tag (protein sequence): (SEQ ID NO: 3) DPKKKRKV. ER tag: (SEQ ID NO: 8) KDEL  Endosome tag: (SEQ ID NO: 9) NPTY--DXXLL--YXXoo (protein sequence; tandem motifs; oo = 2 two residues with hydrophobic side groups; Ciliary tag: VxPx-RVxP-KVHPSST-AxEGG (protein sequence; tandem motifs-SEQ ID NO: 10) Human endosome sequence tag (SEQ ID NO: 11) MTSRKKVLLKVIILGDSGVGKTSLMHRYVND  SEQ ID NO: 16 human synapsin tag: CCTGCAGGGCCCACTAGTATCTGCAGAGGGCCCTGCGTATGAG TGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGC CTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACC CCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGG GAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGC ACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCAC CGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTC CCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGC CGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATA GGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACT CAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTC GTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCG CCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCC CAAGTCGCAGCC SEQ ID NO: 17 Human synaptic vesicle tag: MDCLCIVTTK KYRYQDEDTP PLEHSPAHLP NQANSPPVIV NTDT LEAPGYELQVNGTEGE MEYFEITLER GNSGLGFSIA GGTDNPHIG D DPSIFITKIIPGGAAAQDGR LRVNDSILFV NEVDBREBTH SAAV EALKEA GSIVRLYVMRRKPPAEKVME IKLIKGPKGL GFSIAGGVGN STSLEITASM TempoEndo protein seq; TempoVol with Endosome tag (SEQ ID NO: 13) MTSRKKVLLKVIILGDSGVGKTSLMHRYVNDMSSVRYEQREEPS MVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVHWNDVDQG PNGKSEVRNEERIDIPEISALWWGENEHGADDGRMELPATMWNK LRKVISPFVMSFGERVEGVVLIIVDEVLVIVDLSVTDKSSNATT AIESISLAISFFFLIDIILRIFVEGFNQYFSSKLNIFDAAIVIV TLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIERL ASQKRELRLASRRTISQN. Tags are (in most cases) N-terminal for each sensor-reporter construct.

Example 4 Methods of Reprogramming Cells

Human fibroblasts can be reprogrammed to a pluripotent state. Such cells can then be differentiated to a degree to provide cell type specific model systems for expression of the biosensors.

Fibroblast cells are reprogrammed into inducible pluripotent stem cells (iPSC) over the course of ˜10 days, using a circadian rhythm (cellular synchronization) induced method in combination with transcriptional regulatory control modification. For example, human clock gene and human Bmal1/2/3/4 genes can be employed, e.g., using associated E-box promoters.

Each reprogramming factor is typically a transcriptional regulator that is specific for the cellular lineage and each factor is modified to be controlled by a circadian regulatory element (E-box promoters or an artificial E-box-like promoter sequence tag). The promoter sequences are added to each transcriptional regulator, thus forming a novel transcriptional element and control, regulated by human Clock and Bmal genes.

Fibroblast cells can be reprogrammed into inducible neurons (iN), glial cells (astrocytes included, iG), and inducible neural progenitor cells (iNPCs) using a similar approach as the iPSC but with specific transcription factors suitable for their lineages.

Co-cultures of iN and iG are performed to create a 3D model, using a proprietary in vitro scaffold.

Example 5 Methods for Screening Reprogrammed Cells

The sensors described herein can be introduced into reprogrammed cells, e.g., to monitor interactions of signaling moieties intracellularly. Further, the reprogrammed cells can be exposed to candidate agent molecules to monitor the effects of the agents on intracellular calcium levels and/or voltage potential of the cells.

Nucleic acids encoding the biosensors can be transiently (48 hrs) transduced into selected cells for expression, e.g., using standard electroporation techniques. Further genetic modifications can be performed on the cells to express genes-of-interest (a GPCR, channel, receptor, etc). Such cells can act as representative models for study of signaling interactions and bioactive agents.

In one option, cells are initially directly derived from patient samples. After reprogramming and introduction of biosensors, such cells can provide model systems and assay results more likely to reflect patient responses. In the patient derived cell context, e.g., patient fibroblasts can be rendered pluripotent, differentiated to the extent desired to reflect, e.g., a target cell of a pathology. Depending on the pathology of interest, a biosensor is adapted to be targeted to a location of interest, to detect relevant voltage or calcium levels, and provide a signal appropriate to the detection system in use.

In another option, an external (e.g., non-endogenous) gene of interest is introduced to the cells for study. For example, a gene of interest, such as a receptor molecule, GPCR, or a channel, can be molecularly cloned from a human cDNA library before the gene and transfected into the cells. A biosensor is also introduced to the cells, and a cellular functional assay is performed. In this way, encoded peptides of the gene can be studies in the unique environment of relevant cells. Such model cells (e.g., reprogrammed human cells expressing abnormal receptors) can provide biosensor signals more likely to reflect associated live animal results than other in vitro assays.

The following is a typical experimental design. Cells of interest are seeded into a multi-well format (12, 24, 48, 96, 384, or 1538-well formats). Sensor expression constructs are introduced using standard transfection procedures. 36-48 hours post transfection, cellular functional assays may begin, after correctly assessing the expression levels of the sensors. Stimuli for each experiment depends on the nature of the receptor or channel being assayed and what are the relevant phenotypic/disease conditions applied. If an agent of interest (e.g., a particular molecule or ligand) is known, it is dissolved in liquid solution to be applied to the cells.

A baseline can be established before application of the agent. Typically excitation wavelengths are set to ˜450-570 nm. Emission wavelengths typically range ˜500-700 nm. Fluorescence is sampled every ˜3 seconds at an exposure time ˜500 ms. ΔF/F is calculated by subtracting the baseline fluorescence level (e.g., 20 data points), subtracted from the actual fluorescence response and normalized to the starting fluorescence value. The percent fluorescence change is calculated for each region of interest in a cell or tissue using scripts in ImageJ, Matlab or any proprietary softwares developed by manufacturers (such as Molecular Devices, Tecan, BioTek, etc.) software algorithms.

Example 6 Screening and Discovery Using Human Cellular Models

It is envisioned that the biosensors and model cell systems described herein can be used in any number of formats and contexts to monitor intercellular interactions, signal transduction, and intracellular responses. For example, the following utilities can be realized.

1) Human iPSCs-derived can be employed to cell types to generate 2D or 3D cellular models of human disease models and using biosensors to screen for chemical, biologics, drug-like, and toxicology compounds, e.g., to identify and evaluate candidate drug compounds.

2) A synthesized and genetically encoded biosensor can be prepared to function as a reporter of Ca²⁺ concentrations in human cells, e.g., resolving events in cellular compartments such as the nucleus, cytoplasm, plasma membrane, and/or the like.

3) A synthesized and genetically encoded biosensor can function as a reporter of cellular voltage changes in human cells, e.g., in compartments such as the nucleus, cytoplasm, or plasma membrane. Voltage sensors could evaluate and report fluctuations in membrane potentials due to changes in sodium (Na⁺) and potassium (K⁺) concentrations.

4) Biosensors can be used in a cell-type specific manner. For example, based on the disclosures herein, one can genetically modify a biosensor to contain specific promoter sequences for cell types, e.g., dopaminergic neurons, GABAergic neurons, astrocytes, cardiomyocytes, HSCs, NPCs, MSCs, cancer cells, cancer stem cells, and/or the like. Human and mammalian cell types are generally of particular importance and interest.

5) Biosensors signaling in long wavelength ranges (e.g., 500 nm-750 nm range) can be used to detect interactions through three dimensions of a biologic sample.

6) Using biosensors with sequence identities with at least 70% identity to the following sequences (to be shown soon).

7) Using human cellular models conveniently derived from fibroblast or blood to provide information specific to a patient or cell type.

8) Reprogramming Parkinson patient-derived inducible pluripotent cells (iPSCs) from blood or fibroblast biopsies are into neural progenitor cells (NPCs). Appropriate biosensors are introduced to the cells using electroporation or chemical transfection methods (standard). Plate cells into multi-well plates (24, 48, 95, or 1500-well plates). Apply compounds to the cells via standard loading methods. Fluorescence excitation begins the experiment. Stimulation solutions are followed by washing solutions. Measurements are taken and changes in fluorescence (ΔF) and ratio (ΔF/F) are calculated and plotted against time (milli-seconds to seconds timeframe). Alternatively, to calculate using a single intensiometric reporter would require standardization of the baseline responses and calculation of the changes in fluorescent intensities in response to a chemical or biological compound.

9) Rett syndrome patient-derived inducible pluripotent cells (iPSCs) from blood or fibroblast biopsies are induced into primary neurons, astrocytes and co-cultures of neuronastrocytes from affected-patients and unaffected individuals. Biosensors are introduced to the cells using electroporation or chemical transfection methods (standard). Cells are plated into multi-well plates (24, 48, 95, or 1500-well plates). Compounds are applied to the cells via standard loading methods. Fluorescence excitation begins the experiment. Stimulation solutions are followed by washing solutions. Measurements are taken and changes in fluorescence (ΔF) and ratio (ΔF/F) are calculated and plotted against time (milli-seconds to seconds timeframe). Alternatively, to calculate using a single intensiometric reporter would require standardization of the baseline responses and calculation of the changes in fluorescent intensities in response to a chemical or biological compound.

10) An early-to-mid-stage biopharmaceutical company looking for a new method of screening their library of compounds (specifically, biologics and synthesized peptides) requires a new screening method. Progenitor cells (NPCs, MSCs, HSCs) are generated and voltage-biosensors are introduced to the cells. Then, cells are plated into multi-well plates (24, 48, 95, or 1500-well plates). Compounds are applied to the cells via standard loading methods. Fluorescence excitation begins the experiment. Stimulation solutions are followed by washing solutions. Measurements are taken and changes in fluorescence (ΔF) and ratio (ΔF/F) are calculated and plotted against time (milli-seconds to seconds timeframe). Alternatively, to calculate using a single intensiometric reporter would require standardization of the baseline responses and calculation of the changes in fluorescent intensities in response to a chemical or biological compound.

11) A company wishes to adopt a new toxicology technology to evaluate the effects of their candidate compounds in a physiologically relevant cellular environment, e.g., using human primary/derived/induced cell types and co-cultures in a 2D or 3D model. Equipment would include a multi-plate fluorescent reader (example: TECAN's M1000Pro) or a high-content cellular imager (GE's IN Cell 6000).

12) A company wishes to screen a drug candidate against a population of patients' derived cells (personalized medicine indications), in order to determine the most appropriate genotypes for the candidate compound (for clinical phase 1/2/3 studies and evaluation). The company could be offered two sets of custom-services—first, to make progenitor cell types (NPCs, HPCs, or MSCs) and introduce biosensors for screening; second, to make specific cell types (neurons, glial, cardiomyocytes, liver, etc) and introduce biosensors for screening.

Example 7 2D versus 3D Human Cellular Models

Traditionally, cell culture models in 2D are performed as monolayers. Culturing human cells in 3D requires specialized cell culture dishes with coating (e.g., Lipidure® coating or Nunc®) and sometimes, U-shaped, V-shaped, or F-shaped dish bottoms. Recently, numerous studies in human cancer and stem cell fields have pointed to the importance of 3D culture. 3D cultures allow cancer cells to form tumors as spheroids (see FIG. 21. Many in the academic literature have suggested that the 3D spheroids are more predictive of cancer cellular responses to drug compounds. Thus, using 3D spheroid models provide a new and improved model to predict tumor responses to a chemical compound (aka. chemical compound in a drug library).

Example 8 ATP Biosensor

ATP sensing is important to cellular function, especially in the mitochondria. For mammalian cells, ATP measurements indicate metabolic functions, which may be indicative of dysfunctions due to disease states due to human mutations or polymorphisms, signal transduction pathway disruptions, or mitochondrial dysfunction. Thus, developing an ATP biosensor as a cellular reporter is of critical importance. Tempo's ATP biosensor includes a domain that senses and binds to ATP and a domain that is a fluorescent reporter in the 605 nm to 635 nm (excitation/emission) range.

ATP-binding biosensor, TempoATP:

(SEQ ID NO: 14) MDYKDDDDKKTNWQKRIYRVKPCVICKVAPRDWWVENRHLRIYT MCKTCFSNCINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPD EDRHWSASCHHHHHHMGMSGSMVSKGEELIKENMRMKVVMEGSV NGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDILATSFM YGSRTFIKYPKGIPDEEKQSFPEGFTWERVTRYEDGGVVTVMQD TSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPAD GGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAV DHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDELYK.

Example 9 Heme Biosensor

Heme sensing is important to cellular function, especially in the studies of mitochondria function, as well as in cancer/tumor hypoxia studies (need Refs?). For mammalian cells, cellular metabolic rate is measured by oxygen consumption and indicates metabolic functions. Dysfunctions and irregularities in the cellular models may be due to disease phenotypes in the cell (due to human mutations or polymorphisms), signal transduction pathway disruptions, or mitochondrial dysfunction. Thus, developing a Heme biosensor as a cellular reporter is of critical importance. Tempo's Heme biosensor includes a domain that senses and binds to heme (amino acid residues 1-205) and a domain that is a fluorescent reporter in the 605 nm to 635 nm (excitation/emission) range.

Oxygen-heme-binding biosensor, TempoHEME

(SEQ ID NO: 15) MAAMLEPEPVVAEGTAAQAVETPDWEAPEDAGAQPGSYEIRHYG PAKWVSTCVESMDWDSAVQTGFTKLNSYIQGKNEKGMKIKMTAP VLSYVEPGPGPFSESTITISLYIPSEQQSDPPRPSESDVFIEDR AKMTVFARCFEGFCSAQKNQEQLLTLASILREEGKVFDEKVFYT AGYNSPFRLLDKNNEVWLIQKNKPFKANEMVSKGEELIKENMRM KVVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAF DILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDG GVVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPN TEMMYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNI KMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDEL YK.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes. 

What is claimed is:
 1. A voltage sensor peptide construct comprising: a transmembrane domain adapted to integrate into a membrane of a human cell; voltage sensing domain sensitive to Na⁺, H⁺, or K⁺ concentration; and, fluorescent reporter domain, wherein the fluorescent reporter is adapted to fluoresce at wavelengths in the range from 500 nm to 750 nm; wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to conformational changes in the voltage sensing domain.
 2. The voltage sensor peptide of claim 1, wherein the transmembrane domain and voltage sensing domain are different.
 3. The voltage sensor peptide of claim 1, wherein the voltage sensing domain comprises a sequence at least 80% identical to: (SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKK VHWNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGGDDGRMEVPA TWWNKLRKVISPFVMSFGFRVFGVVLIIVDFVLVIVDLSVTDKSSGAT TAISSISLAISFFFLIDIILHIFVEGFSQYFSSKLNIFDAAIVIVTLL VTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIIILVRILRLA SQKTISQN.


4. The voltage sensor peptide of claim 3, wherein the sequence retains I123, R220, R226, R229, R235, or R238.
 5. The voltage sensor peptide of claim 4, wherein the sequence retains R235 and I123.
 6. The voltage sensor peptide of claim 1, wherein the fluorescent reporter domain comprises a sequence at least 80% identical to: (SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQT AKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGF TWERVTRYEDGGVVTVMQDTSLED.


7. The voltage sensor peptide of claim 1, wherein the construct includes the peptide sequences of both SEQ ID NO: 1 and SEQ ID NO:
 2. 8. A nucleic acid construct encoding the voltage sensor of claim
 1. 9. The nucleic acid construct of claim 8, wherein the nucleic acid construct further comprises a tag sequence selected from the group consisting of: a NLS tag and a ciliary tag.
 10. The nucleic acid construct of claim 9, wherein the NLS tag comprises the peptide sequence: DPKKKRKV (SEQ ID NO: 3)
 11. A human cell comprising the voltage sensor of claim
 1. 12. The cell of claim 11, wherein the human cell is a iPSC derived cell.
 13. The cell of claim 12, wherein the human cell is derived from a fibroblast or a blood cell.
 14. A calcium sensor peptide construct comprising: a troponin EFhand domain; a calcium binding EFhand domain; and, fluorescent reporter domain, wherein the fluorescent reporter is adapted to emit wavelengths in the range from 500 nm to 750 nm; wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to conformational changes in the one or more of the EFhand domains.
 15. The calcium sensor peptide construct of claim 14, wherein the calcium binding domain comprises a calmodulin peptide sequence.
 16. The calcium sensor peptide construct of claim 14, wherein the calcium binding domain is at least 80% identical to: (SEQ ID NO: 12) MVDSSRRKWNKAGHAVRAIGRLSSPVVSFRMYPEDGALKSEIKKGLRL KDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCE RAEGRHSTGGMDELYKGGTGGSLVSKGEENNMAVIKAEFMRFKEHMEA GSVNGHEFEIAEGEGEGRPYEAGTQTARLKVTKGGPLPFAWDAILSPQ IMYGSAKAYVKHPADIAPDYLKLSEPEAGFKWERVMNFEDGGVVHVNQ ADSSLQDGVFIAYKVKLRGTNFAPPDGPVMQKKATMGWEATRDQLTEE EFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSLG QNPTEAELQDMINEVDADGDGTEDEPEFLTMMARKMMNDTDSEEEGVQ GTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIEDLMK DSDKNNGRIDFGEKLTDEEVFKEAFSLEDKDGDGTITTKELGTVMRSL ELDAHEEVDEDGSGTIDFEEFLVMMVRQGQNPTKEEELANCFRIFDKN ADGFIDIEELGEILRAT 


17. The calcium sensor peptide construct of claim 14, wherein the EFhand binding domain is at least 80% identical to: (SEQ ID NO: 12) MVDSSRRKWNKAGHAVRAIGRLSSPVVSERMYPEDGALKSEIKKGLR LKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQ CERAEGRHSTGGMDELYKGGTGGSLVSKGEENNMAVIKAEFMRFKEH MEAGSVNGHEFEIAEGEGEGRPYEAGTQTARLKVTKGGPLPFAWDAI LSPQIMYGSAKAYVKHPADIAPDYLKLSEPEAGFKWERVMNFEDGGV VHVNQADSSLQDGVFIAYKVKLRGTNFAPPDGPVMQKKATMGWEATR DQLTEEEFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELG TVMRSLGQNPTEAELQDMINEVDADGDGTEDEPEFLTMMARKMMNDT DSEEEGVQGTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVT EEDIEDLMKDSDKNNGRIDFGEKLTDEEVFKEAFSLEDKDGDGTITT KELGTVMRSLELDAHEEVDEDGSGTIDFEEFLVMMVRQGQNPTKEEE LANCFRIFDKNADGFIDIEELGEILRAT 


18. The calcium sensor peptide construct of claim 14, wherein the fluorescent reporter domain is at least 90% identical to: (SEQ ID NO: 6) MVKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQ TAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFP EGFTWERVTRYEDGGVVTVMQDTSLED.


19. The calcium sensor peptide construct of claim 14, wherein the construct includes the peptide sequences of both SEQ ID NO: 4 and FKEAFSLFDKDGDGTITTKELGTVMRSL-ELDAIIEEVDEDGSGTIDFEEFLVMMVRQ (SEQ ID NO: 5).
 20. A nucleic acid construct encoding the calcium sensor of claim
 14. 21. The nucleic acid construct of claim 20, further comprising a tag sequence selected from the group consisting of: a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, a mitochondrial tag, a synapsin tag, a cellular compartmental tag, a synaptic vesicle tag, and a ciliary tag.
 22. A human cell comprising the calcium sensor of claim
 14. 23. The cell of claim 22, wherein the human cell is a iPSC derived cell.
 24. The cell of claim 22, wherein the human cell is derived from a fibroblast or a blood cell.
 25. A method of reprogramming fibroblasts, the method comprising: transforming the fibroblasts with one or more constructs comprising a human clock gene and human Bmal1/2/3/4 genes having E-box promoters; synchronizing the circadian rhythm of the fibroblasts; modifying transcriptional regulatory control of the fibroblasts, thereby converting them into inducible pluripotent stem cells; and, reprogramming the stem cells into inducible neurons (iN), glial cells (astrocytes included, iG), or inducible neural progenitor cells (iNPCs).
 26. The method of claim 25, wherein said modifying transcriptional control comprises providing specific transcription factors suitable for a lineage of the fibroblasts.
 27. The method of claim 25, wherein the transcription factor comprises a transcription factor specific for a cellular lineage of the fibroblasts modified with a circadian regulatory element.
 28. The method of claim 27, wherein the transcription factor is selected from the group consisting of: an E-box promoter, an artificial E-box-like promoter sequence tag, and a synthetic transcriptional enhancer element.
 29. The method of claim 25, wherein the transforming construct comprises a nucleic acid encoding the voltage sensor of claim
 1. 30. The method of claim 25, wherein the transforming construct comprises a nucleic acid encoding the calcium sensor of claim
 14. 31. The method of claim 25, further comprising co-culturing iN cells and iG cells to create a 3D model on an in vitro scaffold.
 32. A method for screening active agents, the method comprising: transforming one or more cells of claim 25 with a nucleic acid encoding the voltage sensor or calcium sensor; expressing the voltage sensor or calcium sensor in the cells; contacting the cells with candidate active agents; and, detecting a change in florescence of the voltage sensor or calcium sensor in response to the agent.
 33. The method of claim 32, wherein the agent is selected from the group consisting of: a small molecule chemical library; a ligand to a receptor, a ligand or repressor of a G-protein coupled receptor; a ligand, neurotransmitter; or repressor of an ion channel.
 34. The method of claim 32, further comprising imaging the live cells using low, medium, or high throughput assay formats. 